In recent years, matrix assisted laser desorption ionization (MALDI) mass spectrometry, a technique that provides minimal fragmentation and high sensitivity for the analysis of a wide variety of fragile and non-volatile compounds, has become widely used. MALDI is often combined with time-of-flight (TOF) mass spectrometry, FTICR, quadrupole ion trap, and triple quadrupole mass spectrometers, providing for detection of large molecular masses. This technique can be used to determine molecular weights of biomolecules and their fragment ions, monitor bioreactions, detect post-translational modifications, and perform protein and oligonucleotide sequencing, for tissue imaging, and many more applications.
In its simplest form, the MALDI technique involves depositing the sample (analyte) and a matrix dissolved in a solvent as a spot on a target plate. After the solvent has evaporated, the mixture of sample and matrix is left on the target plate. This is inserted into a mass spectrometer where a pulse from a laser irradiates the matrix and causes it to evaporate. The sample is carried with the matrix, ionized, and analyzed by the mass spectrometer.
Sample preparation methods often involve dilution of small amounts of sample (analyte) in a large molar excess of matrix molecules, typically small organic compounds, in solution. The mixture of matrix and sample is deposited as a spot at a defined target region on a sample plate that may contain as many as 384 or more target regions. As the solvent slowly evaporates, matrix crystals are formed at the target region and may become visible even to the naked eye. The resulting areas of sample deposition can be quite inhomogeneous, with areas of high matrix and sample density and other areas of low or zero density coexisting within a target region. There may also be errors in the positioning of the sample spot at the target region that result in sample spots that are not positioned in the center of the target region.
Once the solvent has evaporated, the sample plate containing the sample spots is inserted into the mass spectrometer and the sample at each target region is analyzed. Typically the diameter of the laser beam where it impacts the target is considerably smaller than the diameter of the sample spot, and data from multiple laser pulses directed at different regions of the sample spot are used to analyze the sample. Sample spot regions can be selected for irradiation with the laser manually, by viewing an image of the sample with a high magnification video system, or automatically by moving the laser or sample plate through a series of predefined positions (such as spiral or zig-zags for example) that cover the target region area that is expected to contain the sample spot.
Manually selecting regions within the sample spot typically requires the full time attention of a skilled operator and is generally not amenable to automation. Automatically moving the laser focal point or the sample plate so that the laser beam focuses on predefined regions within in the sample spot can lead to data sets where the laser pulse has missed the sample completely due to inhomogeneity of the sample spot within the target region. This can result in poor data quality or significantly extended analysis times as the number of laser shots for each target region is increased to ensure that adequate data is acquired.
Some techniques make it possible to resolve inhomogeneous mixtures of matrix and analyte. However these techniques require the precise alignment of the laser of the mass spectrometry apparatus with the samples on the sample plate, such that the laser impinges on the crystals at the points of greatest intensity. This is known hunting for “sweet spots”.
All of the methods described above can be tedious, time-consuming and expensive, generally requiring the services of well trained personnel, the out-sourcing of sample preparation or the need to facilitate sample preparation in-house at considerable expense.